Compositions and methods for reprogramming eukaryotic cells

ABSTRACT

The present invention relates to methods for changing the state of differentiation of a eukaryotic cell, the methods comprising introducing mRNA encoding one or more reprogramming factors into a cell and maintaining the cell under conditions wherein the cell is viable and the mRNA that is introduced into the cell is expressed in sufficient amount and for sufficient time to generate a cell that exhibits a changed state of differentiation compared to the cell into which the mRNA was introduced, and compositions therefor. For example, the present invention provides mRNA molecules and methods for their use to reprogram human somatic cells into pluripotent stem cells.

The present application claims priority to U.S. Provisional ApplicationSer. No. 61/267,312 filed Dec. 7, 2009, which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and rapid, efficientmethods for changing the state of differentiation of a eukaryotic cell.For example, the present invention provides mRNA molecules and methodsfor their use to reprogram cells, such as to reprogram human somaticcells to pluripotent stem cells.

BACKGROUND

In 2006, it was reported (Takahashi and Yamanaka 2006) that theintroduction of genes encoding four protein factors (OCT4 (Octamer-4;POU class 5 homeobox 1), SOX2 (SRY (sex determining region Y)-box 2),KLF4 (Krueppel-like factor 4), and c-MYC) into differentiated mousesomatic cells induced those cells to become pluripotent stem cells,(referred to herein as “induced pluripotent stem cells,” “iPS cells,” or“iPSCs”). Following this original report, pluripotent stem cells werealso induced by transforming human somatic cells with genes encoding thesimilar human protein factors (OCT4, SOX2, KLF4, and c-MYC) (Takahashiet al. 2007), or by transforming human somatic cells with genes encodinghuman OCT4 and SOX2 factors plus genes encoding two other human factors,NANOG and LIN28 (Lin-28 homolog A) (Yu et al. 2007). All of thesemethods used retroviruses or lentiviruses to integrate genes encodingthe reprogramming factors into the genomes of the transformed cells andthe somatic cells were reprogrammed into iPS cells only over a longperiod of time (e.g., in excess of a week).

The generation iPS cells from differentiated somatic cells offers greatpromise as a possible means for treating diseases through celltransplantation. The possibility to generate iPS cells from somaticcells from individual patients also may enable development ofpatient-specific therapies with less risk due to immune rejection. Stillfurther, generation of iPS cells from disease-specific somatic cellsoffers promise as a means to study and develop drugs to treat specificdisease states (Ebert et al. 2009, Lee et al. 2009, Maehr et al. 2009).

Viral delivery of genes encoding protein reprogramming factors (or “iPSCfactors”) provides a highly efficient way to make iPS cells from somaticcells, but the integration of exogenous DNA into the genome, whetherrandom or non-random, creates unpredictable outcomes and can ultimatelylead to cancer (Nakagawa et al. 2008). New reports show that iPS cellscan be created (at lower efficiency) by using other methods that do notrequire genome integration. For example, repeated transfections ofexpression plasmids containing genes for OCT4, SOX2, KLF4 and c-MYC intomouse embryonic fibroblasts to generate iPS cells was demonstrated(Okita et al. 2008). Induced pluripotent stem cells were also generatedfrom human somatic cells by introduction of a plasmid that expressedgenes encoding human OCT4, SOX2, c-MYC, KLF4, NANOG and LIN28 (Yu et al.2009). Other successful approaches for generating iPS cells includetreating somatic cells with: recombinant protein reprogramming factors(Zhou et al. 2009); non-integrating adenoviruses (Stadtfeld et al.2008); or piggyBac transposons (Woltjen et al. 2009) to deliverreprogramming factors. Presently, the generation of iPS cells usingthese non-viral delivery techniques to deliver reprogramming factors isextremely inefficient. Future methods for generating iPS cells forpotential clinical applications will need to increase the speed andefficiency of iPS cell formation while maintaining genome integrity.

SUMMARY OF THE INVENTION

The present invention relates to compositions and rapid, efficientmethods for changing the state of differentiation of a eukaryotic cell.For example, the present invention provides mRNA molecules and methodsfor their use to reprogram cells, such as to reprogram human somaticcells to pluripotent stem cells.

In some embodiments, the present invention provides methods for changingthe state of differentiation of a cell comprising: introducing an mRNAencoding an iPS cell induction factor into a somatic cell to generate areprogrammed cell. In certain embodiments, the introducing comprisesdelivering the mRNA to the somatic cell with a transfection reagent. Inother embodiments, the reprogrammed cell is a dedifferentiated cell. Infurther embodiments, the reprogrammed cell is a transdifferentiatedcell.

In particular embodiments, the mRNA is polyadenylated. In otherembodiments, the mRNA comprises a poly-A tail 100-200 nucleotides inlength. In further embodiments, the mRNA comprises capped mRNA. Incertain embodiments, the mRNA is a population of mRNA molecules, thepopulation having greater than 99% capped mRNA. In additionalembodiments, the mRNA comprises pseudouridine in place of uridine. Inother embodiments, the iPS cell induction factor is selected from thegroup consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. Inparticular embodiments, the introducing comprises introducing mRNAencoding a plurality of iPS cell induction factors into the somaticcell. In further embodiments, the plurality of iPS cell inductionfactors comprises each of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. Inadditional embodiments, the cell is a fibroblast. In other embodiments,the reprogrammed cell is a pluripotent stem cell. In other embodiments,the dedifferentiated cell expresses NANOG and TRA-1-60. In furtherembodiments, the cell is in vitro. In additional embodiments, the cellresides in culture. In particular embodiments, the cells reside inMEF-conditioned medium.

In certain embodiments, the present invention provides compositionscomprising an mRNA encoding an iPS cell induction factor, the mRNAhaving pseudouridine in place of uridine. In other embodiments, thecomposition comprises mRNA encoding a plurality of iPS cell inductionfactors, selected from the group consisting of KLF4, LIN28, c-MYC,NANOG, OCT4, and SOX2. In further embodiments, the plurality comprisesthree or more, or four or more, or five or more, or six.

In certain embodiments, the compositions described above are packaged ina kit. In some embodiments, the compositions comprise a transfectionreagent and an mRNA encoding an iPS cell induction factor.

DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these figures in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows that mRNAs encoding each of the six human reprogrammingfactors, prepared as described in the EXAMPLES, are expressed whentransfected into human newborn 1079 fibroblasts. Phase contrast imagesof the human 1079 fibroblasts which were not transfected with an mRNAencoding a reprogramming factor (i.e., untreated) and which were notstained with a labeled antibody specific for a reprogramming factor areshown in A, E, I, M, Q, and U. Phase contrast images of untreated human1079 fibroblasts which were stained using a labeled antibody specificfor the indicated reprogramming factor show the endogenous expression ofthat respective reprogramming factor protein in B, F, J, N, R, and V.Phase contrast images of the human 1079 fibroblasts which weretransfected with an mRNA encoding the indicated reprogramming factor(i.e., treated or transfected), but which were not stained with alabeled antibody specific for a reprogramming factor are shown in C, G,K, O, S, and W; and the corresponding images of the human 1079fibroblasts that were transfected with mRNA encoding the indicatedreprogramming factor and then stained with respective labeled antibodyspecific for that reprogramming factor 24 hours post-transfection areshown in D, H, L, P, T, and X. A-T are at 20× magnification. U-X are at10× magnification.

FIG. 2 shows that mRNA encoding human reprogramming factors (KLF4,LIN28, c-MYC, NANOG, OCT4, and SOX2) produce iPS cells in human somaticcells. FIG. 2 shows phase contrast images of an iPS cell colony at 12days after the final transfection with mRNA encoding reprogrammingfactors (A, C). NANOG staining (green) is observed in colony #1 (B, D).Images A and B are at 10× magnification. C and D are at 20×magnification.

FIG. 3 shows that iPS colonies derived from human 1079 and IMR90 somaticcells are positive for NANOG and TRA-1-60. FIG. 3 shows phase contrastimages of iPS colonies derived from 1079 cells (A, D) and IMR90 cells(G). The same iPS colony shown in (A) is positive for both NANOG (B) andTRA-1-60 (C). The iPS colony shown in (D) is NANOG-positive (E) andTRA-1-60-positive (F). The iPS colony generated from IMR90 fibroblasts(G) is also positive for both NANOG (H) and TRA-1-60 (I). All images areat 20× magnification.

FIG. 4 shows that rapid, enhanced-efficiency iPSC colony formation isachieved by transfecting cells with mRNA encoding reprogramming factorsin MEF-conditioned medium. Over 200 colonies were detected 3 days afterthe final transfection, in the 10-cm dish transfected three times with36 μg of each reprogramming mRNA (i.e., encoding KLF4, LIN28, c-MYC,NANOG, OCT4, and SOX2). Representative iPSC colonies are shown at 4× (A,B), 10× (C-E) and 20× magnification (F). Eight days after the final mRNAtransfection with mRNAs encoding the six reprogramming factors, greaterthan 1000 iPSC colonies were counted in IMR90 cells transfected with 18μg (G, I) or 36 μg (H) of each of the six mRNAs. Representative coloniesare shown at 4× magnification (G-H) and at 10× magnification (I).

FIG. 5 shows that 1079- and IMR90-derived iPSC colonies are positive forboth NANOG and TRA-1-60 expression. Eight days after the final mRNAtransfection with 36 μg of mRNA for each of the six reprogrammingfactors, the 1079-derived iPSC colonies (shown in A, D, and G) arepositive for NANOG (B, E, and H) and TRA-1-60 (C, F, and I). Eight daysafter the final mRNA transfection with 18 μg (J-L) or 36 μg (M-O) ofmRNA for each of the six reprogramming factors, IMR90-derived iPScolonies are also positive for NANOG (K, N) and TRA-1-60 (L, O).

FIG. 6 provides the mRNA coding sequence for KLF4 (SEQ ID NO:1) andLIN28 (SEQ ID NO:2).

FIG. 7 provides the mRNA coding sequence for cMYC (SEQ ID NO:3) andNANOG (SEQ ID NO:4).

FIG. 8 provides the mRNA coding sequence for OCT4 (SEQ ID NO:5) and SOX2(SEQ ID NO:6).

DEFINITIONS

The present invention will be understood and interpreted based on termsas defined below.

The terms “comprising”, “containing”, “having”, “include”, and“including” are to be construed as “including, but not limited to”unless otherwise noted. The terms “a,” “an,” and “the” and similarreferents in the context of describing the invention and, specifically,in the context of the appended claims, are to be construed to cover boththe singular and the plural unless otherwise noted. The use of any andall examples or exemplary language (“for example”, “e.g.”, “such as”) isintended merely to illustrate aspects or embodiments of the invention,and is not to be construed as limiting the scope thereof, unlessotherwise claimed.

With respect to the use of the word “derived”, such as for an RNA(including mRNA) or a polypeptide that is “derived” from a sample,biological sample, cell, tumor, or the like, it is meant that the RNA orpolypeptide either was present in the sample, biological sample, cell,tumor, or the like, or was made using the RNA in the sample, biologicalsample, cell, tumor, or the like by a process such as an in vitrotranscription reaction, or an RNA amplification reaction, wherein theRNA or polypeptide is either encoded by or a copy of all or a portion ofthe RNA or polypeptide molecules in the original sample, biologicalsample, cell, tumor, or the like. By way of example, such RNA can befrom an in vitro transcription or an RNA amplification reaction, with orwithout cloning of cDNA, rather than being obtained directly from thesample, biological sample, cell, tumor, or the like, so long as theoriginal RNA used for the in vitro transcription or an RNA amplificationreaction was from the sample, biological sample, cell, tumor, or thelike.

The terms “sample” and “biological sample” are used in their broadestsense and encompass samples or specimens obtained from any source thatcontains or may contain eukaryotic cells, including biological andenvironmental sources. As used herein, the term “sample” when used torefer to biological samples obtained from organisms, includes bodilyfluids (e.g., blood or saliva), feces, biopsies, swabs (e.g., buccalswabs), isolated cells, exudates, and the like. The organisms includefungi, plants, animals, and humans. However, these examples are not tobe construed as limiting the types of samples or organisms that find usewith the present invention. In addition, in order to perform research orstudy the results related to use of a method or composition of theinvention, in some embodiments, a “sample” or “biological sample”comprises fixed cells, treated cells, cell lysates, and the like. Insome embodiments, such as embodiments of the method wherein the mRNA isdelivered into a cell from an organism that has a known disease or intoa cell that exhibits a disease state or a known pathology, the “sample”or “biological sample” also comprises bacteria or viruses.

As used herein, the term “incubating” and variants thereof meancontacting one or more components of a reaction with another componentor components, under conditions and for sufficient time such that adesired reaction product is formed.

As used herein, a “nucleoside” refers to a molecule consisting of aguanine (G), adenine (A), thymine (T), uridine (U), pseudouridine(abbreviated by the Greek letter psi-Ψ), or cytidine (C) base covalentlylinked to a pentose sugar, whereas “nucleotide” or “mononucleotide”refers to a nucleoside phosphorylated at one of the hydroxyl groups ofthe pentose sugar.

Linear nucleic acid molecules are said to have a “5′ terminus” (5′ end)and a “3′ terminus” (3′ end) because, except with respect to adenylation(as described elsewhere herein), mononucleotides are joined in onedirection via a phosphodiester linkage to make oligonucleotides, in amanner such that a phosphate on the 5′ carbon of one mononucleotidesugar moiety is joined to an oxygen on the 3′ carbon of the sugar moietyof its neighboring mononucleotide. Therefore, an end of anoligonucleotide is referred to as the “5′ end” if its 5′ phosphate isnot linked to the oxygen of the 3′ carbon of a mononucleotide sugarmoiety, and as the “3′ end” if its 3′ oxygen is not linked to a 5′phosphate of a subsequent mononucleotide sugar moiety. A terminalnucleotide, as used herein, is the nucleotide at the end position of the3′ or 5′ terminus.

In order to accomplish specific goals, a nucleic acid base, sugarmoiety, or internucleoside linkage in one or more of the nucleotides ofthe mRNA that is introduced into a eukaryotic cell in any of the methodsof the invention may comprise a modified base, sugar moiety, orinternucleoside linkage. For example, one or more of the nucleotides ofthe mRNA can have a modified nucleic acid base comprising or consistingof: xanthine; allyamino-uracil; allyamino-thymidine; hypoxanthine;2-aminoadenine; 5-propynyl uracil; 5-propynyl cytosine; 4-thiouracil;6-thioguanine; an aza or deaza uracil; an aza or deaza thymidine; an azaor deaza cytosines; an aza or deaza adenine; or an aza or deazaguanines; or a nucleic acid base that is derivatized with a biotinmoiety, a digoxigenin moiety, a fluorescent or chemiluminescent moiety,a quenching moiety or some other moiety. Still further, one or more ofthe nucleotides of the mRNA can have a sugar moiety, such as, but notlimited to: 2′-fluoro-2′-deoxyribose or 2′-O-methyl-ribose, whichprovide resistance to some nucleases; or 2′-amino-2′-deoxyribose or2′-azido-2′-deoxyribose, which can be labeled by reacting them withvisible, fluorescent, infrared fluorescent or other detectable dyes orchemicals having an electrophilic, photoreactive, alkynyl, or otherreactive chemical moiety. Still further, one or more of the nucleotidesof the mRNA can have a modified internucleoside linkage, such as, butnot limited to, a phosphorothioate, phosphorodithioate,phosphoroselenate, or phosphorodiselenate linkage, which are resistantto some nucleases. The invention is not limited to the modified nucleicacid bases, sugar moieties, or internucleoside linkages listed, but thelist is presented to show examples which may be used for a particularpurpose in a method.

As used herein, a “nucleic acid” or a “polynucleotide” is a covalentlylinked sequence of nucleotides in which the 3′ position of the sugarmoiety of one nucleotide is joined by a phosphodiester bond to the 5′position of the sugar moiety of the next nucleotide (i.e., a 3′ to 5′phosphodiester bond), and in which the nucleotides are linked inspecific sequence; i.e., a linear order of nucleotides. As used herein,an “oligonucleotide” is a short polynucleotide or a portion of apolynucleotide. For example, but without limitation, an oligonucleotidemay be between 10-60 nucleotides in length. In some embodiments, theoligonucleotide consists of or comprises 2′-deoxyribonucleotides (DNA).In some embodiments, the oligonucleotide consists of or comprisesribonucleotides (RNA).

The terms “isolated” or “purified” when used in relation to apolynucleotide or nucleic acid, as in “isolated RNA” or “purified RNA”refers to a nucleic acid that is identified and separated from at leastone contaminant with which it is ordinarily associated in its source.Thus, an isolated or purified nucleic acid (e.g., DNA and RNA) ispresent in a form or setting different from that in which it is found innature, or a form or setting different from that which existed prior tosubjecting it to a treatment or purification method. For example, agiven DNA sequence (e.g., a gene) is found on the host cell chromosometogether with other genes as well as structural and functional proteins,and a specific RNA (e.g., a specific mRNA encoding a specific protein),is found in the cell as a mixture with numerous other RNAs and othercellular components. The isolated or purified polynucleotide or nucleicacid may be present in single-stranded or double-stranded form.

A “cap” or a “cap nucleotide” means a nucleoside-5′-triphosphate that,under suitable reaction conditions, is used as a substrate by a cappingenzyme system and that is thereby joined to the 5′-end of an uncappedRNA comprising primary RNA transcripts or RNA having a 5′-diphosphate.The nucleotide that is so joined to the RNA is also referred to as a“cap nucleotide” herein. A “cap nucleotide” is a guanine nucleotide thatis joined through its 5′ end to the 5′ end of a primary RNA transcript.The RNA that has the cap nucleotide joined to its 5′ end is referred toas “capped RNA” or “capped RNA transcript” or “capped transcript.” Acommon cap nucleoside is 7-methylguanosine or N⁷-methylguanosine(sometimes referred to as “standard cap”), which has a structuredesignated as “m⁷G,” in which case the capped RNA or “m⁷G-capped RNA”has a structure designated as m⁷G(5′)ppp(5′)N₁(pN)_(x)—OH(3′), or moresimply, as m⁷GpppN₁(pN)_(x) or m⁷G[5′]ppp[5′]N, wherein m⁷G representsthe 7-methylguanosine cap nucleoside, ppp represents the triphosphatebridge between the 5′ carbons of the cap nucleoside and the firstnucleotide of the primary RNA transcript, N₁(pN)_(x)—OH(3′) representsthe primary RNA transcript, of which N₁ is the most 5′-nucleotide, “p”represents a phosphate group, “G” represents a guanosine nucleoside,“m⁷” represents the methyl group on the 7-position of guanine, and“[5′]” indicates the position at which the “p” is joined to the riboseof the cap nucleotide and the first nucleoside of the mRNA transcript(“N”). In addition to this “standard cap,” a variety of othernaturally-occurring and synthetic cap analogs are known in the art. RNAthat has any cap nucleotide is referred to as “capped RNA.” The cappedRNA can be naturally occurring from a biological sample or it can beobtained by in vitro capping of RNA that has a 5′ triphosphate group orRNA that has a 5′ diphosphate group with a capping enzyme system (e.g.,vaccinia capping enzyme system or Saccharomyces cerevisiae cappingenzyme system). Alternatively, the capped RNA can be obtained by invitro transcription (IVT) of a DNA template that contains an RNApolymerase promoter, wherein, in addition to the GTP, the IVT reactionalso contains a dinucleotide cap analog (e.g., a m⁷GpppG cap analog oran N⁷-methyl, 2′-O-methyl-GpppG ARCA cap analog or an N⁷-methyl,3′-O-methyl-GpppG ARCA cap analog) using methods known in the art (e.g.,using an AMPLICAP™ T7 capping kit (EPICENTRE)).

Capping of a 5′-triphosphorylated primary mRNA transcript in vivo (orusing a capping enzyme system in vitro) occurs via several enzymaticsteps (Higman et al. 1992, Martin et al. 1975, Myette and Niles 1996).

The following enzymatic reactions are involved in capping of eukaryoticmRNA:

(1) RNA triphosphatase cleaves the 5′-triphosphate of mRNA to adiphosphate,

pppN₁(p)N_(x)—OH(3′)→ppN₁(pN)_(x)—OH(3′)+Pi; and then

(2) RNA guanyltransferase catalyzes joining of GTP to the 5′-diphosphateof the most 5′ nucleotide (N₁) of the mRNA,

ppN₁(pN)_(x)—OH(3′)+GTP→G(5′)ppp(5′)N₁(pN)_(x)—OH(3′)+PPi; and finally,

(3) guanine-7-methyltransferase, using S-adenosyl-methionine (AdoMet) asa co-factor, catalyzes methylation of the 7-nitrogen of guanine in thecap nucleotide,

G(5′)ppp(5′)N₁(pN)_(x)—OH(3′)+AdoMet→m⁷G(5′)ppp(5′)N₁(pN)_(x)—OH(3′)+AdoHyc.

RNA that results from the action of the RNA triphosphatase and the RNAguanyltransferase enzymatic activities, as well as RNA that isadditionally methylated by the guanine-7-methyltransferase enzymaticactivity, is referred to herein as “5′ capped RNA” or “capped RNA”, anda “capping enzyme system” or, more simply, a “capping enzyme” hereinmeans any combination of one or more polypeptides having the enzymaticactivities that result in “capped RNA.” Capping enzyme systems,including cloned forms of such enzymes, have been identified andpurified from many sources and are well known in the art (Banerjee 1980,Higman et al. 1992, Higman et al. 1994, Myette and Niles 1996, Shuman1995, Shuman 2001, Shuman et al. 1980, Wang et al. 1997). Any cappingenzyme system that can convert uncapped RNA that has a 5′ polyphosphateto capped RNA can be used to provide a capped RNA for any of theembodiments of the present invention. In some embodiments, the cappingenzyme system is a poxvirus capping enzyme system. In some preferredembodiments, the capping enzyme system is vaccinia virus capping enzyme.In some embodiments, the capping enzyme system is Saccharomycescerevisiae capping enzyme. Also, in view of the fact that genes encodingRNA triphosphatase, RNA guanyltransferase andguanine-7-methyltransferase from one source can complement deletions inone or all of these genes from another source, the capping enzyme systemcan originate from one source, or one or more of the RNA triphosphatase,RNA guanyltransferase, and/or guanine-7-methyltransferase activities cancomprise a polypeptide from a different source.

A “modified cap nucleotide” of the present invention means a capnucleotide wherein the sugar, the nucleic acid base, or theinternucleoside linkage is chemically modified compared to thecorresponding canonical 7-methylguanosine cap nucleotide. Examples of amodified cap nucleotide include a cap nucleotide comprising: (i) amodified 2′- or 3′-deoxyguanosine-5′-triphosphate (or guanine 2′- or3′-deoxyribonucleic acid-5′-triphosphate) wherein the 2′- or 3′-deoxyposition of the deoxyribose sugar moiety is substituted with a groupcomprising an amino group, an azido group, a fluorine group, a methoxygroup, a thiol (or mercapto) group or a methylthio (or methylmercapto)group; or (ii) a modified guanosine-5′-triphosphate, wherein the O6oxygen of the guanine base is substituted with a methyl group; or (iii)3′-deoxyguanosine. For the sake of clarity, it will be understood hereinthat an “alkoxy-substituted deoxyguanosine-5′-triphosphate” can also bereferred to as an “O-alkyl-substituted guanosine-5′-triphosphate”; byway of example, but without limitation,2′-methoxy-2′-deoxyguanosine-5′-triphosphate (2′-methoxy-2′-dGTP) and3′-methoxy-3′-deoxyguanosine-5′-triphosphate (3′-methoxy-3′-dGTP) canalso be referred to herein as 2′-O-methylguanosine-5′-triphosphate(2′-OMe-GTP) and 3′-O-methylguanosine-5′-triphosphate (3′-OMe-GTP),respectively. Following joining of the modified cap nucleotide to the5′-end of the uncapped RNA comprising primary RNA transcripts (or RNAhaving a 5′-diphosphate), the portion of said modified cap nucleotidethat is joined to the uncapped RNA comprising primary RNA transcripts(or RNA having a 5′-diphosphate) may be referred to herein as a“modified cap nucleoside” (i.e., without referring to the phosphategroups to which it is joined), but sometimes it is referred to as a“modified cap nucleotide”.

A “modified-nucleotide-capped RNA” is a capped RNA molecule that issynthesized using a capping enzyme system and a modified cap nucleotide,wherein the cap nucleotide on its 5′ terminus comprises the modified capnucleotide, or a capped RNA that is synthesize co-transcriptionally inan in vitro transcription reaction that contains a modified dinucleotidecap analog wherein the dinucleotide cap analog contains the chemicalmodification in the cap nucleotide. In some embodiments, the modifieddinucleotide cap analog is an anti-reverse cap analog or ARCA (Grudzienet al. 2004, Grudzien-Nogalska et al. 2007, Jemielity et al. 2003, Penget al. 2002, Stepinski et al. 2001).

A “primary RNA” or “primary RNA transcript” means an RNA molecule thatis synthesized by an RNA polymerase in vivo or in vitro and which RNAmolecule has a triphosphate on the 5′-carbon of its most 5′ nucleotide.

An “RNA amplification reaction” or an “RNA amplification method” means amethod for increasing the amount of RNA corresponding to one or multipledesired RNA sequences in a sample. For example, in some embodiments, theRNA amplification method comprises: (a) synthesizing first-strand cDNAcomplementary to the one or more desired RNA molecules by RNA-dependentDNA polymerase extension of one or more primers that anneal to thedesired RNA molecules; (b) synthesizing double-stranded cDNA from thefirst-strand cDNA using a process wherein a functional RNA polymerasepromoter is joined thereto; and (c) contacting the double-stranded cDNAwith an RNA polymerase that binds to said promoter under transcriptionconditions whereby RNA corresponding to the one or more desired RNAmolecules is obtained. Unless otherwise stated related to a specificembodiment of the invention, an RNA amplification reaction according tothe present invention means a sense RNA amplification reaction, meaningan RNA amplification reaction that synthesizes sense RNA (e.g., RNAhaving the same sequence as an mRNA or other primary RNA transcript,rather than the complement of that sequence). Sense RNA amplificationreactions known in the art, which are encompassed within this definitioninclude, but are not limited to, the methods which synthesize sense RNAdescribed in Ozawa et al. (Ozawa et al. 2006) and in U.S. PatentApplication Nos. 20090053775; 20050153333; 20030186237; 20040197802; and20040171041. The RNA amplification method described in U.S. PatentApplication No. 20090053775 is a preferred method for obtainingamplified RNA derived from one or more cells, which amplified RNA isthen used to make mRNA for use in the methods of the present invention.

A “poly-A polymerase” (“PAP”) means a template-independent RNApolymerase found in most eukaryotes, prokaryotes, and eukaryotic virusesthat selectively uses ATP to incorporate AMP residues to 3′-hydroxylatedends of RNA. Since PAP enzymes that have been studied from plants,animals, bacteria and viruses all catalyze the same overall reaction(Edmonds 1990) are highly conserved structurally (Gershon 2000) and lackintrinsic specificity for particular sequences or sizes of RNA moleculesif the PAP is separated from proteins that recognize AAUAAApolyadenylation signals (Wilusz and Shenk 1988), purified wild-type andrecombinant PAP enzymes from any of a variety of sources can be used forthe present invention.

A “reprogramming factor” means a protein, peptide, or other biomoleculethat, when used alone or in combination with other factors orconditions, causes a change in the state of differentiation of a cell inwhich the reprogramming factor is introduced or expressed. In somepreferred embodiments of the methods of the present invention, thereprogramming factor is a protein or peptide that is encoded by an mRNAthat is introduced into a cell, thereby generating a cell that exhibitsa changed state of differentiation compared to the cell in which themRNA was introduced. In some preferred embodiments of the methods of thepresent invention, the reprogramming factor is a transcription factor.One embodiment of a reprogramming factor used in a method of the presentinvention is an “iPS induction factor.”

An “iPS cell induction factor” is a protein, peptide, or otherbiomolecule that, when used alone or in combination with otherdedifferentiation factors, causes the generation of iPS cells fromsomatic cells. Examples of iPS cell induction factors include OCT4,SOX2, c-MYC, KLF4, NANOG and LIN28. iPS cell induction factors includefull length polypeptide sequences or biologically active fragmentsthereof. Likewise an mRNA encoding an iPS cell induction factor mayencode a full length polypeptide or biologically active fragmentsthereof. The mRNA coding sequence for exemplary iPS induction factorsare shown in FIGS. 6 (KLF4 and LIN28), 7 (cMYC and NANOG), and 8 (OCT4and SOX2). In certain embodiments, the present invention employs thesequences or similar sequences shown in these figures, including mRNAmolecules that additionally comprise, joined to these mRNA sequences,oligoribonucleotides which exhibit any of the 5′ and 3′ UTR sequences,Kozak sequences, IRES sequences, cap nucleotides, and/or poly(A)sequences used in the experiments described herein, or which aregenerally known in the art and which can be used in place of those usedherein by joining them to these protein-coding mRNA sequences for thepurpose of optimizing translation of the respective mRNA molecules inthe cells and improving their stability in the cell in order toaccomplish the methods described herein.

“Differentiation” or “cellular differentiation” means the process bywhich a cell that exhibits a less specialized state of differentiationor cell type becomes a cell that exhibits a more specialized state ofdifferentiation or cell type. Scientists, including biologists, cellbiologists, immunologists, and embryologists, use a variety of methodsand criteria to define, describe, or categorize different cellsaccording to their “cell type,” “differentiated state,” or “state ofdifferentiation.” In general, a cell is defined, described, orcategorized with respect to its “cell type,” “differentiated state,” or“state of differentiation” based on one or more phenotypes exhibited bythat cell, which phenotypes can include shape, a biochemical ormetabolic activity or function, the presence of certain biomolecules inthe cell (e.g., based on stains that react with specific biomolecules),or on the cell (e.g., based on binding of one or more antibodies thatreact with specific biomolecules on the cell surface). For example, insome embodiments, different cell types are identified and sorted using acell sorter or fluorescent-activated cell sorter (FACS) instrument.

“Dedifferentiation” means the process by which a cell that exhibits amore specialized state of differentiation or cell type becomes a cellthat exhibits a less specialized state of differentiation or cell type.For example, in some preferred embodiments of the method of the presentinvention, a differentiated somatic cell (e.g., a mammalian fibroblast)is dedifferentiated into an iPS cell, meaning that the somatic cellloses the more specialized state of differentiation and becomes an iPScell that exhibits a less specialized state of differentiation.

DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods forreprogramming somatic cells to pluripotent stem cells. For example, thepresent invention provides mRNA molecules and their use to reprogramhuman somatic cells into pluripotent stem cells.

Experiments conducted during the development of embodiments of thepresent invention demonstrated that mRNA molecules can be administeredto cells and induce a dedifferentiation process to generatededifferentiated cells—including pluripotent stem cells. Thus, thepresent invention provides compositions and methods for generating iPScells. Surprisingly, the administration of mRNA can provide highlyefficient generation of iPS cells.

In some embodiments, the present invention provides methods fordedifferentiating a somatic cell comprising: introducing mRNA encodingone or more iPSC induction factors into a somatic cell to generate adedifferentiated cell.

In some embodiments, the present invention provides methods fordedifferentiating a somatic cell comprising: introducing mRNA encodingone or more iPSC induction factors into a somatic cell and maintainingthe cell under conditions wherein the cell is viable and the mRNA thatis introduced into the cell is expressed in sufficient amount and forsufficient time to generate a dedifferentiated cell. In some preferredembodiments, the dedifferentiated cell is an induced pluripotent stemcell (iPSC).

In some embodiments, the present invention provides methods for changingthe state of differentiation (or differentiated state) of a eukaryoticcell comprising: introducing mRNA encoding one or more reprogrammingfactors into a cell and maintaining the cell under conditions whereinthe cell is viable and the mRNA that is introduced into the cell isexpressed in sufficient amount and for sufficient time to generate acell that exhibits a changed state of differentiation compared to thecell into which the mRNA was introduced.

In some embodiments, the present invention provides methods for changingthe state of differentiation of a eukaryotic cell comprising:introducing mRNA encoding one or more reprogramming factors into a celland maintaining the cell under conditions wherein the cell is viable andthe mRNA that is introduced into the cell is expressed in sufficientamount and for sufficient time to generate a cell that exhibits achanged state of differentiation compared to the cell into which themRNA was introduced. In some embodiments, the changed state ofdifferentiation is a dedifferentiated state of differentiation comparedto the cell into which the mRNA was introduced. For example, in someembodiments, the cell that exhibits the changed state of differentiationis a pluripotent stem cell that is dedifferentiated compared to asomatic cell into which the mRNA was introduced (e.g., a somatic cellthat is differentiated into a fibroblast, a cardomyocyte, or anotherdifferentiated cell type). In some embodiments, the cell into which themRNA is introduced is a somatic cell of one lineage, phenotype, orfunction, and the cell that exhibits the changed state ofdifferentiation is a somatic cell that exhibits a lineage, phenotype, orfunction that is different than that of the cell into which the mRNA wasintroduced; thus, in these embodiments, the method results intransdifferentiation (Graf and Enver 2009).

The methods of the invention are not limited with respect to aparticular cell into which the mRNA is introduced. In some embodimentsof any of the above methods, the cell into which the mRNA is introducedis derived from any multi-cellular eukaryote. In some embodiments of anyof the above methods, the cell into which the mRNA is introduced isselected from among a human cell, an animal cell, a plant cell, and afungal cell. In some embodiments of any of the above methods, the cellinto which the mRNA is introduced is a normal cell that is from anorganism that is free of a known disease. In some embodiments of any ofthe above methods, the cell into which the mRNA is introduced is a cellfrom an organism that has a known disease. In some embodiments of any ofthe above methods, the cell into which the mRNA is introduced is a cellthat is free of a known pathology. In some embodiments of any of theabove methods, the cell into which the mRNA is introduced is a cell thatexhibits a disease state or a known pathology (e.g., a cancer cell, or apancreatic beta cell that exhibits metabolic properties characteristicof a diabetic cell).

The invention is not limited to the use of a specific cell type (e.g.,to a specific somatic cell type) in embodiments of the methodscomprising introducing mRNA encoding one or more iPSC cell inductionfactors in order to generate a dedifferentiated cell (e.g., an iPScell). Any cell that is subject to dedifferentiation using iPS cellinduction factors is contemplated. Such cells include, but are notlimited to, fibroblasts, keratinocytes, adipocytes, lymphocytes,T-cells, B-Cells, cells in mononuclear cord blood, buccal mucosa cells,hepatic cells, HeLa, MCF-7 or other cancer cells. In some embodiments,the cells reside in vitro (e.g., in culture) or in vivo. In someembodiments, when generated in culture, a cell-free conditioned medium(e.g., MEF-conditioned medium) is used. As demonstrated below, such amedium provided enhanced iPS cell generation. The invention is notlimited, however, to the culturing conditions used. Any culturingcondition or medium now known or later identified as useful for themethods of the invention (e.g., to generate iPS cells from somatic cellsand maintain said cells) is contemplated for use with the invention. Forexample, although not preferred, in some embodiments of the method, afeeder cell layer is used instead of conditioned medium for culturingthe cells that are treated using the method.

In some embodiments of any of these methods, the step of introducingmRNA comprises delivering the mRNA into the cell (e.g., a human or otheranimal somatic cell) with a transfection reagent (e.g., TRANSIT™ mRNAtransfection reagent, MirusBio, Madison, Wis.). However, the inventionis not limited by the nature of the transfection method utilized.Indeed, any transfection process known, or identified in the future thatis able to deliver mRNA molecules into cells in vitro or in vivo, iscontemplated, including methods that deliver the mRNA into cells inculture or in a life-supporting medium, whether said cells compriseisolated cells or cells comprising a eukaryotic tissue or organ, ormethods that deliver the mRNA in vivo into cells in an organism, such asa human, animal, plant or fungus. In some embodiments, the transfectionreagent comprises a lipid (e.g., liposomes, micelles, etc.). In someembodiments, the transfection reagent comprises a nanoparticle ornanotube. In some embodiments, the transfection reagent comprises acationic compound (e.g., polyethylene imine or PEI). In someembodiments, the transfection method uses an electric current to deliverthe mRNA into the cell (e.g., by electroporation).

The data presented herein shows that, with respect to the mRNAintroduced into the cell, certain amounts of the mRNAs used in theEXAMPLES described herein resulted in higher efficiency and more rapidinduction of pluripotent stem cells from the particular somatic cellsused than other amounts of mRNA. However, the methods of the presentinvention are not limited to the use of a specific amount of mRNA tointroduce into the cell. For example, in some embodiments, a total ofthree doses, with each dose comprising 18 micrograms of each of sixdifferent mRNAs, each encoding a different human reprogramming factor,was used to introduce the mRNA into approximately 3×10⁵ human fibroblastcells in a 10-cm plate (e.g., delivered using a lipid-containingtransfection reagent), although in other embodiments, higher or loweramounts of the mRNAs were used to introduce into the cells.

The invention is not limited to a particular chemical form of the mRNAused, although certain forms of mRNA may produce more efficient results.In some embodiments, the mRNA is polyadenylated. For example, in somepreferred embodiments, the mRNA comprises a poly-A tail (e.g., a poly-Atail having 50-200 nucleotides, e.g., preferably 100-200, 150-200nucleotides, or greater than 150 nucleotides), although in someembodiments, a longer or a shorter poly-A tail is used. In someembodiments, the mRNA used in the methods is capped. To maximizeefficiency of expression in the cells, it is preferred that the majorityof mRNA molecules contain a cap. In some preferred embodiments, the mRNAmolecules used in the methods are synthesized in vitro by incubatinguncapped primary RNA in the presence of with a capping enzyme system. Insome preferred embodiments, the primary RNA used in the capping enzymereaction is synthesized by in vitro transcription (IVT) of a DNAmolecule that encodes the RNA to be synthesized. The DNA that encodesthe RNA to be synthesized is joined to an RNA polymerase promoter, towhich, an RNA polymerase binds and initiates transcription therefrom.The IVT can be performed using any RNA polymerase so long as synthesisof the template that encodes the RNA is specifically and sufficientlyinitiated from a respective cognate RNA polymerase promoter. In somepreferred embodiments, the RNA polymerase is selected from among T7 RNApolymerase, SP6 RNA polymerase and T3 RNA polymerase. In some otherembodiments, capped RNA is synthesized co-transcriptionally by using adinucleotide cap analog in the IVT reaction (e.g., using an AMPLICAP™ T7Kit; EPICENTRE Technologies Corporation, Madison, Wis.). However, use ofa separate IVT reaction, followed by capping with a capping enzymesystem, which results in approximately 100% of the RNA being capped, ispreferred over co-transcriptional capping, which typically results inonly about 80% of the RNA being capped. Thus, in some preferredembodiments, a high percentage of the mRNA molecules used in a method ofthe present invention are capped (e.g., greater than 80%, greater than90%, greater than 95%, greater than 98%, greater than 99%, greater than99.5%, or greater than 99.9% of the population of mRNA molecules arecapped). In some preferred embodiments, the mRNA used in the methods ofthe present invention has a cap with a cap1 structure, meaning that thepenultimate nucleotide with respect to the cap nucleotide has a methylgroup on the 2′-position of the ribose. However, in some embodiments,mRNA used in the methods has a cap with a cap0 structure, meaning thatthe penultimate nucleotide with respect to the cap nucleotide does nothave a methyl group on the 2′-position of the ribose. With some but notall transcripts, transfection of eukaryotic cells with mRNA having a capwith a cap1 structure results in a higher level or longer duration ofprotein expression in the transfected cells compared to transfection ofthe same cells with the same mRNA but with a cap having a cap0structure. In some embodiments, the mRNA used in the methods of thepresent invention has a modified cap nucleotide. In some experimentsperformed prior to the experiments presented in the EXAMPLES herein, thepresent Applicants found that, when 1079 or IMR90 human fibroblast cellswere transfected with OCT4 mRNA that contained either uridine orpseudouridine in place of uridine, the pseudouridine-containing mRNA wasexpressed at a higher level or for a longer duration than the mRNA thatcontained uridine. Therefore, in some preferred embodiments, one or moreor all of the uridines contained in the mRNA(s) used in the methods ofthe present invention is/are replaced by pseudouridine (e.g., bysubstituting pseudouridine-5′-triphosphate in the IVT reaction tosynthesize the RNA in place of uridine-5′-triphosphate). However, insome embodiments, the mRNA used in the methods of the invention containsuridine and does not contain pseudouridine. In addition, in order toaccomplish specific goals, a nucleic acid base, sugar moiety, orinternucleoside linkage in one or more of the nucleotides of the mRNAthat is introduced into a eukaryotic cell in any of the methods of theinvention may comprise a modified nucleic acid base, sugar moiety, orinternucleoside linkage.

The invention is also not limited with respect to the source of the mRNAthat is delivered into the eukaryotic cell in any of the methods of theinvention. In some embodiments, such as those described in the EXAMPLES,the mRNA is synthesized in vitro by transcription of a DNA templatecomprising a gene cloned in a linearized plasmid vector or a PCR orRT-PCR amplification product, capping using a capping enzyme system, andpolyadenylation using a poly-A polymerase. In some other embodiments,the mRNA that is delivered into the eukaryotic cell in any of themethods of the invention is derived directly from a cell or a biologicalsample. For example, in some embodiments, the mRNA derived from a cellor biological sample is obtained by amplifying the mRNA from the cell orbiological sample using an RNA amplification reaction.

With respect to the methods comprising introducing mRNA encoding one ormore iPSC cell induction factors in order to generate a dedifferentiatedcell (e.g., an iPS cell), the invention is not limited by the nature ofthe iPS cell induction factors used. Any mRNA encoding one or moreprotein induction factors now known, or later discovered, that find usein dedifferentiation, are contemplated for use in the present invention.In some embodiments, one or more mRNAs encoding for KLF4, LIN28, c-MYC,NANOG, OCT4, or SOX2 are employed. Oct-3/4 and certain members of theSox gene family (Sox1, Sox2, Sox3, and Sox15) have been identified astranscriptional regulators involved in the induction process. Additionalgenes, however, including certain members of the Klf family (Klf1, Klf2,Klf4, and Klf5), the Myc family (C-myc, L-myc, and N-myc), Nanog, andLIN28, have been identified to increase the induction efficiency. Anyone or more such factors may be used as desired.

While the compositions and methods of the invention may be used togenerated iPS cells, the invention is not limited to the generation ofsuch cells. For example, in some embodiments, mRNA encoding one or morereprogramming factors is introduced into a cell in order to generate acell with a changed state of differentiation compared to the cell intowhich the mRNA was introduced. For example, in some embodiments, mRNAencoding one or more iPS cell induction factors is used to generate adedifferentiated cell that is not an iPS cells. Such cells find use inresearch, drug screening, and other applications.

In some embodiments, the present invention further provides methodsemploying the dedifferentiated cells generated by the above methods. Forexample, such cells find use in research, drug screening, andtherapeutic applications in humans or other animals. For example, insome embodiments, the cells generated find use in the identification andcharacterization of iPS cell induction factors as well as other factorsassociated with differentiation or dedifferentiation. In someembodiments, the generated dedifferentiated cells are transplanted intoan organism or into a tissue residing in vitro or in vivo. In someembodiments, an organism, tissue, or culture system housing thegenerated cells is exposed to a test compound and the effect of the testcompound on the cells or on the organism, tissue, or culture system isobserved or measured.

In some other embodiments, a dedifferentiated cell generated using theabove methods (e.g., an iPS cell) is further treated to generate adifferentiated cell that has the same state of differentiation or celltype compared to the somatic cell from which the dedifferentiated cellwas generated. In some other embodiments, the dedifferentiated cellgenerated using the above methods (e.g., an iPS cell) is further treatedto generate a differentiated cell that has a different state ofdifferentiation or cell type compared to the somatic cell from which thededifferentiated cell was generated. In some embodiments, thedifferentiated cell is generated from the generated dedifferentiatedcell (e.g., the generated iPS cell) by introducing mRNA encoding one ormore reprogramming factors into the generated iPS cell and maintainingthe cell into which the mRNA is introduced under conditions wherein thecell is viable and is differentiated into a cell that has a changedstate of differentiation or cell type compared to the generateddedifferentiated cell (e.g., the generated iPS cell) into which the mRNAencoding the one or more reprogramming factors is introduced. In some ofthese embodiments, the generated differentiated cell that has thechanged state of differentiation is used for research, drug screening,or therapeutic applications (e.g., in humans or other animals). Forexample, the generated differentiated cells find use in theidentification and characterization of reprogramming factors associatedwith differentiation. In some embodiments, the generated differentiatedcells are transplanted into an organism or into a tissue residing invitro or in vivo. In some embodiments, an organism, tissue, or culturesystem housing the generated differentiated cells is exposed to a testcompound and the effect of the test compound on the cells or on theorganism, tissue, or culture system is observed or measured.

In some preferred embodiments of the method comprising introducing mRNAencoding one or more iPSC induction factors into a somatic cell andmaintaining the cell under conditions wherein the cell is viable and themRNA that is introduced into the cell is expressed in sufficient amountand for sufficient time to generate a dedifferentiated cell (e.g.,wherein the dedifferentiated cell is an induced pluripotent stem cell),the sufficient time to generate a dedifferentiated cell is less than oneweek. In some preferred embodiments of this method, the reprogrammingefficiency for generating dedifferentiated cells is greater than orequal to 50 dedifferentiated cells (e.g., iPSCs) per 3×10⁵ input cellsinto which the mRNA is introduced. In some preferred embodiments of thismethod, the reprogramming efficiency for generating dedifferentiatedcells is greater than or equal to 100 dedifferentiated cells (e.g.,iPSCs) per 3×10⁵ input cells into which the mRNA is introduced. In somepreferred embodiments of this method, the reprogramming efficiency forgenerating dedifferentiated cells is greater than or equal to 150dedifferentiated cells (e.g., iPSCs) per 3×10⁵ input cells into whichthe mRNA is introduced. In some preferred embodiments of this method,the reprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 200 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 300 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 400 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 500 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 600 dedifferentiated cells per 3×10⁵ inputcells (e.g., iPSCs) into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 700 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 800 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. In some preferredembodiments of this method, the reprogramming efficiency for generatingdedifferentiated cells is greater than or equal to 900 dedifferentiatedcells (e.g., iPSCs) per 3×10⁵ input cells into which the mRNA isintroduced. In some preferred embodiments of this method, thereprogramming efficiency for generating dedifferentiated cells isgreater than or equal to 1000 dedifferentiated cells (e.g., iPSCs) per3×10⁵ input cells into which the mRNA is introduced. Thus, in somepreferred embodiments, this method was greater than 2-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 5-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 10-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 20-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 25-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 30-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 35-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).In some preferred embodiments, this method was greater than 40-fold moreefficient than the published protocol comprising delivery ofreprogramming factors with a viral vector (e.g., a lentivirus vector).

The present invention further provides compositions (systems, kits,reaction mixtures, cells, mRNA) used or useful in the methods and/orgenerated by the methods described herein. For example, in someembodiments, the present invention provides an mRNA encoding an iPS cellinduction factor, the mRNA having pseudouridine in place of uridine.

The present invention further provides compositions comprising atransfection reagent and an mRNA encoding an iPS cell induction factor(e.g., a mixture of transfection reagent and mRNA).

In some embodiments, the compositions comprise mRNA encoding a plurality(e.g., 2 or more, 3 or more, 4 or more, 5 or more, or 6) of iPS cellinduction factors, including, but not limited to, KLF4, LIN28, c-MYC,NANOG, OCT4, and SOX2.

The compositions may further comprise any other reagent or componentsufficient, necessary, or useful for practicing any of the methodsdescribed herein. Such reagents or components include, but are notlimited to, transfection reagents, culture medium (e.g., MEF-conditionmedium), cells (e.g., somatic cells, iPS cells), containers, boxes,buffers, inhibitors (e.g., RNase inhibitors), labels (e.g., fluorescent,luminescent, radioactive, etc.), positive and/or negative controlmolecules, reagents for generating capped mRNA, dry ice or otherrefrigerants, instructions for use, cell culture equipment,detection/analysis equipment, and the like.

In certain embodiments, the mRNAs are purified into purified RNApreparations that have most of the contaminating RNA molecules removed(e.g., molecules that cause an immunogenic response in the cells), suchas described in U.S. application Ser. No. 12/962,468 filed Dec. 7, 2010,which is herein incorporated by reference.

EXAMPLES

The following experimental protocols were employed in the examplesprovided below, unless indicated otherwise.

Cell Culture. Newborn human foreskin fibroblast 1079 cells (Cat#CRL-2097, ATCC, Manassas, Va.) and human IMR90 cells (Cat# CCL-186,ATCC) were cultured in Advanced MEM Medium (Invitrogen, Carlsbad,Calif.) supplemented with 10% heat-inactivated fetal bovine serum (FBS,Hyclone Laboratories, Logan, Utah), 2 mM Glutamax (Invitrogen), 0.1 mMβ-mercaptoethanol(Sigma, St. Louis, Mo.), and Penicillin/Streptomycin(Invitrogen). All cells were grown at 37° C. and 5% CO₂. In someexperiments, human iPS cells that were induced using methods describedherein were maintained on irradiated mouse embryonic fibroblasts (MEFs)(R&D Systems, Minneapolis, Minn.) on 10-cm plates pre-coated with 0.1%gelatin (Millipore, Phillipsburg, N.J.) in DMEM/F12 medium supplementedwith 20% KnockOut serum replacer, 0.1 mM L-glutamine (all fromInvitrogen), 0.1 mM β-mercaptoethanol (Sigma) and 100 ng/ml basicfibroblast growth factor (Invitrogen). In some experiments, human iPScells that were induced using methods described herein were maintainedin MEF-conditioned medium that had been collected as previouslydescribed (Xu et al. 2001).

Constructions of Vectors. The cDNAs for the open reading frames (ORFs)of KLF4, LIN28, NANOG, and OCT4 were PCR amplified from cDNA clones(Open Biosystems, Huntsville, Ala.), cloned into a plasmid vectordownstream of a T7 RNA polymerase promoter (Mackie 1988, Studier andMoffatt 1986) (e.g., various pBluescript™, Agilent, La Jolla, Calif. orpGEM™, Promega, Madison, Wis., vectors) and sequenced. The ORF of SOX2was PCR amplified from a cDNA clone (Invitrogen) and the ORF of c-MYCwas isolated by RT-PCR from HeLa cell total RNA. Both SOX2 and c-MYC ORFwere also cloned into a plasmid vector downstream of a T7 RNA polymerasepromoter and sequenced.

Alternative plasmid vectors containing human open reading frames of(KLF4, LIN28, c-MYC, NANOG, OCT4 and SOX2) were cloned intopBluescriptII. These pBluescriptII vectors where constructed by ligatingthe above open reading frames into the EcoRV (cMyc) or EcoRV/SpeI (KLF4,LIN28, NANOG, OCT4, and SOX2) sites between the 5′ and 3′ Xenopus laevisB-globin untranslated regions described (Krieg and Melton 1984).

mRNA Production. The T7 RNA polymerase promoter-containing plasmidcontructs (pT7-KLF4, pT7-LIN28, pT7-c-MYC, pT7-OCT4, pT7-SOX2, orpT7-XBg-KLF4, pT7-XBg-LIN28, pT7-XBg-c-MYC, pT7-XBg-OCT4, andpT7-XBg-SOX2) were linearized with BamHI and pT7-NANOG and pT7-XBg-NANOGwere linearized with Xba I. The mSCRIPT™ mRNA production system(EPICENTRE Technologies Corporation, Madison, Wis.) was used to producemRNA with a 5′ Cap1 structure and a 3′ Poly (A) tail (e.g., withapproximately 150 A residues), except that pseudouridine-5′-triphosphate(TRILINK, San Diego, Calif.) was used in place ofuridine-5′-triphosphate in the T7 RNA polymerase in vitro transcriptionreactions.

Reprogramming of Human Somatic Cells on MEFs. 1079 fibroblasts wereplated at 1×10⁵ cells/well of a 6-well dish pre-coated with 0.1% gelatin(Millipore) and grown overnight. The 1079 fibroblasts were transfectedwith equal amounts of each reprogramming factor mRNA (KLF4, LIN28,c-MYC, NANOG, OCT4, and SOX2) using TransIT mRNA transfection reagent(MirusBio, Madison, Wis.). A total of three transfections wereperformed, with one transfection being performed every other day, withmedia changes the day after the first and second transfection. The dayafter the third transfection, the cells were trypsinized and 3.3×10⁵cells were plated in 1079 medium onto 0.1% gelatin pre-coated 10-cmplate seeded with 7.5×10⁵ MEFs the day before. The day after plating thetransfected 1079 fibroblasts onto MEFs, the medium was changed to iPScell medium. The iPS cell medium was changed every day. Eight days afterplating the transfected cells onto MEFs, MEF-conditioned medium wasused. MEF conditioned medium was collected as previously described (Xuet al. 2001). Plates were screened every day for the presence ofcolonies with an iPS morphology using an inverted microscope.

Alternative protocols for reprogramming 1079 and IMR90 fibroblasts onMEFs were also used. MEFs were plated at 1.25×10⁵ cells/well of a 0.1%gelatin pre-coated 6 well dish and incubated overnight in completefibroblast media. 1079 or IMR90 fibroblasts were plated at 3×104cells/well of a 6 well dish seeded with MEFs the previous day and grownovernight at 37° C./5% CO₂. The mScript Kit was then used to generateCap1/poly-adenylated mRNA from the following vectors (pT7-Xβg-KLF4,pT7-Xβg-LIN28, pT7-Xβg-c-MYC, pT7-Xβg-NANOG, pT7-Xβg-OCT4, andpT7-Xβg-SOX2) for use in these daily transfections. All sixreprogramming mRNAs were diluted to 100 ng/μl of each mRNA. Equalmolarity of each mRNA was added together using the following conversionfactors (OCT4 is set at 1 and all of the other mRNAs are multiplied bythese conversion factors to obtain equal molarity in each mRNA mix).KLF=1.32, LIN28=0.58, c-MYC=1.26, NANOG=0.85, OCT4=1, and SOX2=0.88. Toobtain equal molarity of each factor 132 μl of KLF4, 58 μl of LIN28, 126μl of c-MYC, 85 μl of NANOG, 100 μl of OCT4 and 88 μl of SOX2 mRNA (eachat 100 ng/μl) would be added together. A 600 μg total dose fortransfections would mean that 100 ng (using molarity conversions above)of each of six reprogramming mRNAs was used. Trans-IT mRNA transfectionreagent was used to transfect these mRNA doses. For all transfections,mRNA pools were added to 250 μl of either DMEM/F12 media withoutadditives or Advanced MEM media without additives. 5 μl of mRNA boostreagent and 5 μl of TransIT transfection reagent was added to each tubeand incubated at room temp for two minutes before adding thetransfection mix to 2.5 mls of either Advanced MEM media with 10%FBS+100 ng/ml of hFGFb or iPS media containing 100 ng/ml of hFGFb.Transfections were repeated everyday for 10-16 days. The media waschanged 4 hours after each transfection. In some experiments, the cellswere trypsinized and replated onto new MEF plates between 5-8 days afterthe inititial transfection. 1079 cells were split ⅙ or 1/12 onto new MEFplates while IMR90 cells were split ⅓ or ⅙ onto new MEF plates.

Reprogramming of Human Somatic Cells in MEF-Conditioned Medium. 1079 orIMR90 fibroblasts were plated at 3×10⁵ cells per 10 cm dishes pre-coatedwith 0.1% gelatin (Millipore) and grown overnight. The 1079 or IMR90fibroblasts were transfected with equal amounts of reprogramming factormRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) using TransIT mRNAtransfection reagent (MirusBio, Madison, Wis.). For each transfection,either 6 μg, 18 μg, or 36 μg of each reprogramming mRNA (KLF4, LIN28,c-MYC, NANOG, OCT4, and SOX2) was used per 10-cm dish. A total of threetransfections were performed, with one transfection being performedevery other day with the medium being changed the day after each of thefirst and second transfections. All transfections were performed inMEF-conditioned medium. The day after the third transfection, the cellswere trypsinized and 3×10⁵ cells were plated on new 10-cm dishespre-coated with 0.1% gelatin (Millipore). The cells were grown inMEF-conditioned medium for the duration of the experiment.

Similar daily mRNA transfections were also performed as described in theprevious section with the only difference being that MEFs were not usedas feeder layers, only MEF conditioned media was used.

Immunoflourescence. The 1079 cells or 1079-derived iPS cell plates werewashed with PBS and fixed in 4% paraformaldehyde in PBS for 30 minutesat room temperature. The iPS cells were then washed 3 times for 5minutes each wash with PBS followed by three washes in PBS+0.1% TritonX-100. The iPS cells were then blocked in blocking buffer (PBS+0.1%Triton, 2% FBS, and 1% BSA) for 1 hour at room temperature. The cellswere then incubated for 2 hours at room temperature with the primaryantibody (mouse anti-human OCT4 Cat# sc-5279, Santa Cruz Biotechnology,Santa Cruz, Calif.), (rabbit anti-human NANOG Cat #3580, rabbitanti-human KLF4 Cat # 4038, mouse anti-human LIN28 Cat# 5930, rabbitanti-human c-MYC Cat# 5605, rabbit anti-human SOX2 Cat# 3579, and mouseanti-TRA-1-60 all from Cell Signaling Technology, Beverly, Mass.) at a1:500 dilution in blocking buffer. After washing 5 times in PBS+0.1%Triton X-100, the iPS cells were incubated for 2 hours with theanti-rabbit Alexa Fluor 488 antibody (Cat # 4412, Cell SignalingTechnology), anti-mouse FITC secondary (Cat# F5262, Sigma), or ananti-mouse Alexa Fluor 555 (Cat# 4409, Cell Signaling Technology) at1:1000 dilutions in blocking buffer. Images were taken on a Nikon TS100Finverted microscope (Nikon, Tokyo, Japan) with a 2-megapixel monochromedigital camera (Nikon) using NIS-elements software (Nikon).

Example 1

This example describes tests to determine if transfections with mRNAencoding KLF4, LIN28, c-MYC, NANOG, OCT4 and SOX2 resulted in expressionand proper subcellular localization of each respective protein productin newborn fetal foreskin 1079 fibroblasts. The mRNAs used in theexperiments were made with pseudouridine-5′-triphosphate substitutingfor uridine-5′-triphosphate (Kariko et al. 2008). The 1079 fibroblastswere transfected with 4 μg of each mRNA per well of a 6-well dish andimmunofluorescence analysis was performed 24 hours post-transfection.Endogenous KLF4, LIN28, NANOG, OCT4 and SOX2 protein levels wereundetectable by immunoflourescence in untransfected 1079 cells (FIG. 1B,F, N, R, V). Endogenous levels of c-MYC were relatively high inuntransfected 1079 cells (FIG. 1J). Transfections with mRNAs encodingthe transcription factors, KLF4, c-MYC, NANOG, OCT4, and SOX2 allresulted in primarily nuclear localization of each protein 24 hoursafter mRNA transfections (FIG. 1D, L, P, T, X). The cytoplasmic mRNAbinding protein, LIN28, was localized to the cytoplasm (FIG. 1H).

Example 2

Having demonstrated efficient mRNA transfection and proper subcellularlocalization of the reprogramming proteins, this example describesdevelopment of a protocol for iPS cell generation from somaticfibroblasts. Equal amounts (by weight) of KLF4, LIN28, c-MYC, NANOG,OCT4, and SOX2 mRNAs were transfected into 1079 fibroblasts three times(once every other day). The day after the third transfection, the cellswere plated onto irradiated MEF feeder cells and grown in iPS cellmedium. Six days after plating the 1079 fibroblasts onto irradiatedMEFs, two putative iPS cell colonies became apparent on the 10-cm platetransfected with 3 μg of each reprogramming factor mRNA (KLF4, LIN28,c-MYC, NANOG, OCT4, and SOX2). The colonies were allowed to grow until12 days after the last transfection before they were fixed forimmunofluorescence analysis. The inner cell mass-specific marker NANOGis often used to assay whether iPS cell colonies are truly iPS colonies(Gonzalez et al. 2009, Huangfu et al. 2008). NANOG expression arisingfrom the mRNAs that were transfected 12 days earlier would be negligiblebased on previous reports on the duration of mRNA stability andexpression (Kariko et al. 2008). Staining for NANOG showed that both ofthe two iPS cell colonies were NANOG positive (FIG. 2 B, D, and notshown). The surrounding fibroblasts that were not part of the iPS cellcolony were NANOG negative, suggesting that they were not reprogrammedinto iPS cells.

In a subsequent experiment using the same protocol, both 1079fibroblasts and human IMR90 fibroblasts were transfected with the samereprogramming mRNAs. Multiple colonies were detected as early as 4 daysafter plating the transfected cells on irradiated MEFs. When 6 μg ofeach mRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) were used intransfections in 6-well dishes, 3 putative iPS cell colonies were laterdetected in both cell lines after plating on MEFs in 10-cm plates (FIG.3). In addition to analyzing these colonies for expression of NANOG,TRA-1-60, a more stringent marker of fully reprogrammed iPS cells (Chanet al. 2009), was also used for immunofluorescence analysis. iPScolonies generated from 1079 fibroblasts (FIG. 3 A-F) and from IMR90fibroblasts (FIG. 3 G-I) were positive for both NANOG and TRA-1-60,indicating that these colonies are fully reprogrammed type III iPS cellcolonies. This protocol comprising three transfections of mRNAs encodingall six reprogramming factors and then plating onto MEF feeder cellsresulted in a similar reprogramming efficiency (3-6 iPS colonies per1×10⁶ input cells) as was previously reported by protocols comprisingdelivery of the same reprogramming factors by transfection of anexpression plasmid (Aoi et al. 2008).

Example 3

This example describes attempts to improve the efficiency ofreprogramming differentiated cells using mRNA. In one approach, aprotocol was used that comprised transfecting 1079 or IMR90 fibroblaststhree times (once every other day) with the mRNAs encoding the sixreprogramming factors in MEF-conditioned medium rather than infibroblast medium and then growing the treated 1079 fibroblasts inMEF-conditioned medium rather than plating them on a MEF feeder layerafter the treatments. At the highest transfection dose utilized (36 μgof each reprogramming factor per 10-cm dish), 208 iPS cell colonies weredetected three days after the final transfection (FIG. A-F).Interestingly no iPS cell colonies were detected in the dishestransfected with either 6 or 18 μg of each of the reprogramming factorsat the 3-day timepoint, suggesting that a dose above 18 μg wasimportant, under these conditions, for iPS cell colony formation tooccur within 3 days in MEF-conditioned medium. IMR90 cells showed aneven higher number of iPS cell colonies, with around 200 colonies 8 daysafter the last transfection in the plate transfected with three 6-μgdoses of each of the six reprogramming factor mRNAs and >1000 coloniesin IMR90 cells transfected three times with 18-μg or 36-μg doses of eachof the six reprogramming mRNAs (FIG. 4 G-I). Colonies were visible 3days after the final transfection in 1079 cells, whereas colonies onlybecame visible 6-7 days after the final transfection in IMR90 cells.Therefore, the more mature colonies derived from the 1079 cells werelarger and denser and were darker in brightfield images compared to theIMR90 colonies (FIG. 4). All of the colonies on the 1079 platetransfected three times with 36 μg of each reprogramming mRNA werepositive for both NANOG and TRA-1-60 8 days after the final mRNAtransfection (FIG. 5 A-I). All of the more immature IMR90 iPS colonieswere also positive for both NANOG and TRA-1-60 (FIG. 5 J-O), but showedless robust staining for both markers due to their less dense cellularnature compared to the more mature 1079 colonies (FIG. 5 A-I). Thepresent protocol comprising delivery of the mRNAs into 1079 or IMR90cells in MEF-conditioned medium had a reprogramming efficiency of 200to >1000 colonies per 3×10⁵ input cells. This protocol for inducing iPScells was faster and almost 2-3 orders of magnitude more efficient thanpublished protocols comprising transfecting fibroblasts with DNAplasmids encoding these same six reprogramming factors in fibroblastmedium (Aoi et al. 2008). Still further, this protocol was over 7-40times more efficient than the published protocol comprising delivery ofreprogramming factors with lentiviruses, based on the published datathat lentiviral delivery of reprogramming factors into 1079 newbornfibroblasts, which resulted in approximately 57 iPS cell colonies per6×10⁵ input cells (Aoi et al. 2008). This protocol is also much fasterthan the published methods.

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1. A method for changing the state of differentiation of a somatic cell comprising: introducing an mRNA encoding an iPS cell induction factor into a somatic cell to generate a reprogrammed cell.
 2. The method of claim 1, wherein said introducing comprises delivering said mRNA to said somatic cell with a transfection reagent.
 3. The method of claim 1, wherein said reprogrammed cell is a dedifferentiated cell.
 4. The method of claim 1, wherein said reprogrammed cell is a transdifferentiated cell.
 5. The method of claim 1, wherein said mRNA is polyadenylated.
 6. The method of claim 6, wherein said mRNA comprises a poly-A tail 100-200 nucleotides in length.
 7. The method of claim 1, wherein said mRNA comprises capped mRNA.
 8. The method of claim 1, wherein said mRNA is a population of mRNA molecules, said population having greater than 99% capped mRNA.
 9. The method of claim 1, wherein said mRNA comprises pseudouridine in place of uridine.
 10. The method of claim 1, wherein said iPS cell induction factor is selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.
 11. The method of claim 1, wherein said introducing comprises introducing mRNA encoding a plurality of iPS cell induction factors into said somatic cell.
 12. The method of claim 11, wherein said plurality of iPS cell induction factors comprises each of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.
 13. The method of claim 1, wherein said somatic cell is a fibroblast.
 14. The method of claim 1, wherein said reprogrammed cell is a pluripotent stem cell.
 15. The method of claim 1, wherein said dedifferentiated cell expresses NANOG and TRA-1-60.
 16. The method of claim 1, wherein said cell is in vitro.
 17. The method of claim 1, wherein said cell resides in culture.
 18. The method of claim 17, wherein said cells reside in MEF-conditioned medium.
 19. A composition comprising an mRNA encoding an iPS cell induction factor, said mRNA having pseudouridine in place of uridine.
 20. The composition of claim 19, wherein said composition comprises mRNA encoding a plurality of iPS cell induction factors, selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.
 21. The composition of claim 20, wherein said plurality comprises three or more.
 22. The composition of claim 20, wherein said plurality comprises four or more.
 23. The composition of claim 20, wherein said plurality comprises five or more.
 24. The composition of claim 20, wherein said plurality comprises each of said iPS cell induction factors. 